Hydrogen/deuterium Exchange Monitored By Mass Spectrometry Research Articles

Exploring the conformational landscape of the rabies virus glycoprotein using HDX-MS.

Rabies virus, a member of the lyssavirus family, causes on the order of 60,000 deaths per year, disproportionately affecting historically exploited regions of the world. While prophylactic measures such as vaccines and immunoglobulin therapies are very effective at preventing rabies upon exposure, these treatments are very expensive and require cold-chain logistics, making them inaccessible to much of the world. Furthermore, there is no cure for rabies after the onset of symptoms, making it a death sentence for people who do not seek treatment because either they lack access or they do not suspect the animal they were bitten by was infectious. The rabies virus glycoprotein (RABV-G) is densely displayed on the surface of the virion, making it an attractive target for vaccine and drug development, yet this protein remains poorly understood. RABV-G is a highly dynamic protein that is known to reversibly sample its pre- and post-fusion conformations in equilibrium at physiological pH, making it difficult to study using many in vitro methods. Hydrogen deuterium exchange monitored by mass spectrometry (HDX-MS) is an ideal technique to probe the conformational landscape of proteins that occupy more than one conformation in solution. This work uses HDX-MS to measure the local stability of the protein when biased to both the prefusion and postfusion conformation as well as quantify the kinetics of the conformational change between the two conformations. This work will be useful in exploring how antibodies interact with the different conformations of rabies G in order to design better therapeutic strategies to combat this lethal virus.

Negative allosteric modulation of the glucagon receptor by RAMP2.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The glucagon receptor (GCGR), a Family B GPCR responsible for maintenance of proper blood sugar levels, interacts with RAMP2, though the purpose and consequence of this interaction is poorly understood. Using a series of biochemical and cell-based assays, we show that RAMP2 interacts with and broadly inhibits GCGR-induced downstream signaling. Hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) demonstrate that RAMP2 enhances local dynamics in select locations in and near the receptor extracellular domain (ECD) as well as at a key region in the 6th transmembrane helix, while single-molecule fluorescence resonance energy transfer (smFRET) experiments show that this RAMP2-induced ECD disorder results in inhibition of active and intermediate states of the intracellular face of the receptor. Using cryo-electron microscopy (cryoEM), we determined the structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner, yet the ECD of GCGR is indeed largely disordered in the presence of RAMP2. This disorder is accompanied by rearrangements of several key areas of the receptor, resulting in the formation of a likely unproductive complex. Together, our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H+, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H+ retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H+ allowed the extraction of HDX data from all five glycosylated regions of the serpin α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H+ provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H+ for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.

Avoiding H/D Scrambling with Minimal Ion Transmission Loss for HDX-MS/MS-ETD Analysis on a High-Resolution Q-TOF Mass Spectrometer.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) enables the study of protein dynamics by measuring the time-resolved deuterium incorporation into a protein incubated in D2O. Using electron-based fragmentation in the gas phase it is possible to measure deuterium uptake at single-residue resolution. However, a prerequisite for this approach is that the solution-phase labeling is conserved in the gas phase prior to precursor fragmentation. It is therefore essential to reduce or even avoid intramolecular hydrogen/deuterium migration, which causes randomization of the deuterium labels along the peptide (hydrogen scrambling). Here, we describe an optimization strategy for reducing scrambling to a negligible level while minimizing the impact on sensitivity on a high-resolution Q-TOF equipped with ETD and an electrospray ionization interface consisting of a glass transfer capillary followed by a dual ion funnel. In our strategy we narrowed down the optimization to two accelerating potentials, and we defined the optimization of these in a simple rule by accounting for their interdependency in relation to scrambling and transmission efficiency. Using this rule, we were able to reduce scrambling from 75% to below 5% on average using the highly scrambling-sensitive quadruply charged P1 peptide scrambling probe resulting in a minor 33% transmission loss. To demonstrate the applicability of this approach, we probe the dynamics of certain regions in cytochrome c.

A Dynamic Core in Human NQO1 Controls the Functional and Stability Effects of Ligand Binding and Their Communication across the Enzyme Dimer.

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is a multi-functional protein whose alteration is associated with cancer, Parkinson’s and Alzheimer´s diseases. NQO1 displays a remarkable functional chemistry, capable of binding different functional ligands that modulate its activity, stability and interaction with proteins and nucleic acids. Our understanding of this functional chemistry is limited by the difficulty of obtaining structural and dynamic information on many of these states. Herein, we have used hydrogen/deuterium exchange monitored by mass spectrometry (HDXMS) to investigate the structural dynamics of NQO1 in three ligation states: without ligands (NQO1apo), with FAD (NQO1holo) and with FAD and the inhibitor dicoumarol (NQO1dic). We show that NQO1apo has a minimally stable folded core holding the protein dimer, with FAD and dicoumarol binding sites populating binding non-competent conformations. Binding of FAD significantly decreases protein dynamics and stabilizes the FAD and dicoumarol binding sites as well as the monomer:monomer interface. Dicoumarol binding further stabilizes all three functional sites, a result not previously anticipated by available crystallographic models. Our work provides an experimental perspective into the communication of stability effects through the NQO1 dimer, which is valuable for understanding at the molecular level the effects of disease-associated variants, post-translational modifications and ligand binding cooperativity in NQO1.

Thiol-ene Microfluidic Chip for Performing Hydrogen/Deuterium Exchange of Proteins at Subsecond Time Scales.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become a routine approach for sensitive analysis of the dynamic structure and interactions of proteins. However, transient conformational changes and weak affinity interactions found in many biological systems typically only perturb fast-exchanging amides in proteins. Detection of HDX changes for such amides require shorter deuterium labeling times (subsecond) than can be performed reproducibly by manual sample handling. Here, we describe the development and validation of a microfluidic chip capable of rapid on-chip protein labeling and reaction quenching. The fastHDX thiol-ene microchip is fabricated entirely using thiol-ene photochemistry. The chip has a three-channel design for introduction of protein sample, deuterated buffer, and quench buffer. Thiol-ene based monolith plugs (i.e., polymerized thiol-ene emulsions) situated within microchannels are generated in situ using a 3D-printed photolithography mask. We show that efficient on-chip mixing can be achieved at channel junctions by spatially confined in-channel monolith mixers. Using human hemoglobin (Hb), we demonstrate the ability of the chip to perform highly reproducible HDX in the 0.14-1.1 s time frame. The HDX of Hb at 0.14-1.1 s, resolved to peptide segments, correlates closely with structural features of the crystal structure of the Hb tetramer, with helices exhibiting no or minor HDX and loops undergoing pronounced HDX even at subsecond time scales. On-chip HDX of Hb at time points ranging from 0.14-1.1 s demonstrates the ability to distinguish fast exchanging amides and thus provides enhanced detection of transient structure and interactions in dynamic or exposed regions of proteins in solution.

SEC-SAXS and HDX-MS: A powerful combination. The case of the calcium-binding domain of a bacterial toxin.

Small-angle X-ray scattering (SAXS) is a relatively simple experimental technique that provides information on the global conformation of macromolecules in solution, be they fully structured, partially, or extensively unfolded. Size exclusion chromatography in line with a SAXS measuring cell considerably improves the monodispersity and ideality of solutions, the two main requirements of a "good" SAXS sample. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) offers a wealth of information regarding the solvent accessibility at the local (peptide) level. It constitutes a sensitive probe of local flexibility and, more generally, of structural dynamics. The combination of both approaches presented here is very powerful, as illustrated by the case of RD, a calcium-binding protein that is part of a bacterial virulence factor.

Measuring the hydrogen/deuterium exchange of proteins at high spatial resolution by mass spectrometry: overcoming gas-phase hydrogen/deuterium scrambling.

Proteins are dynamic molecules that exhibit conformational flexibility to function properly. Well-known examples of this are allosteric regulation of protein activity and ligand-induced conformational changes in protein receptors. Detailed knowledge of the conformational properties of proteins is therefore pertinent to both basic and applied research, including drug development, since the majority of drugs target protein receptors and a growing number of drugs introduced to the market are therapeutic peptides or proteins. X-ray crystallography provides a static picture at atomic resolution of the lowest-energy structure of the native ensemble. There is a growing need for sensitive analytical tools to explore all of the significant molecular structures in the conformational landscape of proteins. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has recently emerged as a powerful method for characterizing protein conformational dynamics. The basis of this method is the fact that backbone amides in stable hydrogen-bonded structures (e.g., α-helices and β-sheets) are protected against exchange with the aqueous solvent. All protein structures are dynamic, however, and eventually all of the protecting hydrogen bonds will transiently break as the protein--according to thermodynamic principles--cycles through partially unfolded states that correspond to excited free energy levels. As a result, all of the backbone amides will eventually become temporarily solvent-exposed and exchange-competent over time. Consequently, a folded protein in D2O will gradually incorporate deuterium into its backbone amides, and the kinetics of the process can be readily monitored by mass spectrometry. The deuterium uptake kinetics for the intact protein (global exchange kinetics) represents the sum of the exchange kinetics for the individual backbone amides. Local exchange kinetics is typically achieved by using pepsin digestion under quench conditions (i.e., under cold acidic conditions where the amide hydrogen exchange rate is slowed by many orders of magnitude). The ability to localize the individual deuterated residues (the spatial resolution) is determined by the size (typically ∼7-15 residues) and the number of peptic peptides. These peptides provide a relatively coarse-grained picture of the protein dynamics. A fundamental understanding of the relationship between protein function/dysfunction and conformational dynamics requires in many cases higher resolution and ultimately single-residue resolution. In this Account, we summarize our efforts to achieve single-residue deuterium levels in proteins by electron-based or laser-induced gas-phase fragmentation methods. A crucial analytical requirement for this approach is that the pattern of deuterium labeling from solution is retained in the gas-phase fragment ions. It is therefore essential to control and minimize any occurrence of gas-phase randomization of the solution deuterium label (H/D scrambling) during the MS experiment. For this purpose, we have developed model peptide probes to accurately measure the onset and extent of H/D scrambling. Our analytical procedures to control the occurrence of H/D scrambling are detailed along with the physical parameters that induce it during MS analysis. In light of the growing use of gas-phase dissociation experiments to measure the HDX of proteins in order to obtain a detailed characterization and understanding of the dynamic conformations and interactions of proteins at the molecular level, we discuss the perspectives and challenges of future high-resolution HDX-MS methodology.

Ion source parameters and hydrogen scrambling in the ECD of selectively deuterated peptides

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study protein dynamics in solution. Recently, electron-based fragmentation methods (ECD and ETD) have been utilized in HDX-MS/MS experiments as experimental tools to increase the spatial resolution (the ability to obtain deuterium levels of individual residues). An essential prerequisite for this approach is that the level of hydrogen scrambling is negligible. The occurrence of hydrogen scrambling depends critically on the extent of vibrational excitation in the mass spectrometer. In particular, the desolvation process in the electrospray ion source is likely to induce scrambling at standard operating conditions. Consequently, finding experimental conditions that minimize hydrogen scrambling to a negligible level is thus pivotal for the application of electron-based fragmentation in HDX-MS/MS experiments. In the present work, we investigate the occurrence of scrambling in the Apollo I electrospray ion source using ECD of selectively deuterium labeled peptides. The electrospray ion source settings leading to minimal scrambling were identified. Furthermore, an energy dependent loss of deuterium occurring in the ion source was also observed. This loss was critically dependent on the occurrence of scrambling.

Electrochemical Reduction of Disulfide-Containing Proteins for Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

Characterization of disulfide bond-containing proteins by hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) requires reduction of the disulfide bonds under acidic and cold conditions, where the amide hydrogen exchange reaction is quenched (pH 2.5, 0 °C). The reduction typically requires a high concentration (>200 mM) of the chemical reducing agent Tris(2-carboxyethyl)phosphine (TCEP) as its reduction rate constant is decreased at low pH and temperature. Serious adverse effects on chromatographic and mass spectrometric performances have been reported when using high concentrations of TCEP. In the present study, we explore the feasibility of using electrochemical reduction as a substitute for TCEP in HDX-MS analyses. Our results demonstrate that efficient disulfide bond reduction is readily achieved by implementing an electrochemical cell into the HDX-MS workflow. We also identify some challenges in using electrochemical reduction in HDX-MS analyses and provide possible conditions to attenuate these limitations. For example, high salt concentrations hamper disulfide bond reduction, necessitating additional dilution of the sample with aqueous acidic solution at quench conditions.

Characterizing the Dynamics of α-Synuclein Oligomers Using Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

Soluble oligomers formed by α-synuclein (αSN) are suspected to play a central role in neuronal cell death during Parkinson's disease. While studies have probed the surface structure of these oligomers, little is known about the backbone dynamics of αSN when they form soluble oligomers. Using hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS), we have analyzed the structural dynamics of soluble αSN oligomers. The analyzed oligomers were metastable, slowly dissociating to monomers over a period of 21 days, after excess monomer had been removed. The C-terminal region of αSN (residues 94-140) underwent isotopic exchange very rapidly, demonstrating a highly dynamic region in the oligomeric state. Three regions (residues 4-17, 39-54, and 70-89) were strongly protected against isotopic exchange in the oligomers, indicating the presence of a stable hydrogen-bonded or solvent-shielded structure. The protected regions were interspersed by two somewhat more dynamic regions (residues 18-38 and 55-70). In the oligomeric state, the isotopic exchange pattern of the region of residues 35-95 of αSN corresponded well with previous nuclear magnetic resonance and electron paramagnetic resonance analyses performed on αSN fibrils and indicated a possible zipperlike maturation mechanism for αSN aggregates. We find the protected N-terminus (residues 4-17) to be of particular interest, as this region has previously been observed to be highly dynamic for both monomeric and fibrillar αSN. This region has mainly been described in relation to membrane binding of αSN, and structuring may be important in relation to disease.

Investigation of amide hydrogen back-exchange in Asp and His repeats measured by hydrogen (1H/2H) exchange mass spectrometry

Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry (HDX-MS) has become a valuable tool in studies of protein dynamics and protein interactions. Isotopic exchange is typically initiated by diluting a protein solution into deuterated buffer at physiological conditions. For MS analysis, the exchange reaction is quenched by acidification and cooling and the labeled protein (or a digest thereof) is analyzed by mass spectrometry. An inevitable deuterium loss occurs during quench conditions (i.e., back-exchange). The primary structure of the peptide influences the back-exchange rate due to steric hindrance by bulky side groups and by inductive and charge effects. Here we show that the back-exchange in histidine repeats (His6) at HDX-MS quench conditions is complete within a few seconds using either acetic acid or formic acid in the quench solution, while aspartic acid repeats (Asp6) retain deuterons for several minutes using formic acid. We employ electron transfer dissociation to obtain residue-specific deuterium levels of the Asp repeat in K2D6IIKIIK using a hybrid linear ion trap-Orbitrap mass spectrometer. Our results show an unexpected uneven distribution of deuterium in the Asp repeat. The implication of the rapid back-exchange of His repeats for HDX-MS protein hydrogen exchange studies is discussed. We also discuss the implications of retained deuterons in the Asp repeat of K2D6IIKIIK when this peptide is used as a probe for the occurrence of hydrogen scrambling.

Hydrogen/Deuterium Exchange Mass Spectrometric Analysis of Conformational Changes Accompanying the Assembly of the Yeast Prion Ure2p into Protein Fibrils

The Ure2 protein from baker's yeast ( Saccharomyces cerevisiae) has prion properties. In vitro, at neutral pH, soluble Ure2p forms long, twisted fibrils. Two models have been proposed to account for Ure2p polymerization. The first postulates that a segment of 70 amino acid residues in the flexible N-terminal domain from different Ure2p molecules forms a parallel superpleated β-structure running along the fibrils. The second hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and intramolecular interactions. The knowledge of the three-dimensional structure of the fibrillar form of Ure2p is critical for understanding the molecular events leading to the polymerization of soluble Ure2p into fibrils and hence for the design of inhibitors that might have therapeutic potential as yeast prions possessing domains rich in N and Q residues, similar to huntingtin. Solvent-accessibility studies using hydrogen/deuterium exchange monitored by mass spectrometry (HXMS) can provide insights into the structure of the fibrillar form of Ure2p and characterize at the molecular level the conformational rearrangements that occur upon assembly, in particular through the identification of protected regions and their localization in the overall structure of the protein. We have analyzed the changes in Ure2p structure associated with its assembly into fibrils using HXMS. The deuterium incorporation profile along the sequence allows the identification of the regions that exhibit the most important conformational change. Our data reveal that Ure2p undergoes minor structural changes upon assembly. While polypeptides [82-92] and [13-37] exhibit significant increased and decreased exposure to the solvent, respectively, no marked change was observed for the rest of the protein upon assembly. Our results afford new insights into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] element in yeast.